Simultaneous staining of exponentially growing versus plateau phase cells with the proliferation-associated antibody Ki-67 and propidium iodide: analysis by flow cytometry

Abstract. The antibody Ki-67, which detects proliferating cells, was used in combination with propidium iodide, a DNA-specific dye. The double-staining method allowed discrimination of cells in the phases of the cell cycle as well as the recognition of Ki-67 staining characteristics. Suspension cultures of U937 cells were measured in exponential growth and plateau phase in nutritional deprivation. The fraction of Ki-67 positive cells was nearly 100% 2 days after dilution and 46% 7 days after dilution of the cultures. Stathmokinetic measurements with colchicine and flow cytometry measurements with the BrdU-Hoechst technique yielded close to 100% proliferation at day 2 but only 18% and 6%, respectively, at day 7. The discrepancy between Ki-67 results and the results of the two other methods is considered to be a characteristic of nutritionally deprived cells.     

Gamma-glutamyltransferase,possible novel biomarker in colon diverticulosis: a case-control study

The gamma-glutamyltransferase (GGT) is recognized in medical practice as a useful indicator for the detection of liver lesions, especially those induced by the excessive consumption of alcoholic or cholesterol-associated drinks. The present study, although it includes a very small number of cases diagnosed with colon diverticulosis-diverticulitis associated with polyposis at the same intestinal level, identifies the presence of increased circulating concentrations of this enzyme in the serum. Its serum levels are tracked “dynamically” throughout a year after the diagnosis and start of the therapy. The study calls into question the release of the enzyme from the edge of the enterocytes’ brush-like edge, leading to the pathogenic disturbance of regional redox homeostasis. The hypothesis gives the circulating values of GGT predictive value for cellular oxidative stress, as well as for indirectly expressing the glutathione level in cytosol.     

The organotellurium compound ammonium trichloro(dioxoethylene-o,o')tellurate reacts with homocysteine to form homocystine and decreases homocysteine levels in hyperhomocysteinemic mice

Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and antineurodegenerative activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels.     

Leishmania donovani P23 protects parasites against HSP90 inhibitor-mediated growth arrest

In Leishmania donovani, the HSP90 chaperone complex plays an essential role in the control of the parasite’s life cycle, general viability and infectivity. Several of the associated co-chaperones were also shown to be essential for viability and/or infectivity to mammalian cells. Here, we identify and describe the co-chaperone P23 and distinguish its function from that of the structurally related small heat shock protein HSP23. P23 is expressed constitutively and associates itself with members of the HSP90 complex, i.e. HSP90 and Sti1. Viable P23 gene replacement mutants could be raised and confirmed as null mutants without deleterious effects on viability under a variety of physiological growth conditions. The null mutant also displays near-wild-type infectivity, arguing against a decisive role played by P23 in laboratory settings. However, the P23 null mutant displays a marked hypersensitivity against HSP90 inhibitors geldanamycin and radicicol. P23 also appears to affect the radicicol resistance of a HSP90 Leu₃₃-Ile mutant described previously. Therefore, the annotation of L. donovani P23 as HSP90-interacting co-chaperone is confirmed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0595-y) contains supplementary material, which is available to authorized users.     

Multi locus analysis of Pristionchus pacificus on La Réunion Island reveals an evolutionary history shaped by multiple introductions, constrained dispersal events and rare out-crossing

Pristionchus pacificus, recently established as a model organism in evolutionary biology, is a cosmopolitan nematode that has a necromenic association with scarab beetles. The diverse array of host beetle species and habitat types occupied by P. pacificus make it a good model for investigating local adaptation to novel environments. Presence of P. pacificus on La Réunion Island, a young volcanic island with a dynamic geological history and a wide variety of ecozones, facilitates such investigation in an island biogeographic setting. Microsatellite data from 20 markers and 223 strains and mitochondrial sequence data from 272 strains reveal rich genetic diversity among La Réunion P. pacificus isolates, shaped by differentially timed introductions from diverse sources and in association with different beetle species. Distinctions between volcanic zones and between arid western and wet eastern climatic zones have likely limited westward dispersal of recently colonized lineages and maintained a genetic distinction between eastern and western clades. The highly selfing lifestyle of P. pacificus contributes to the strong fine‐scale population structure detected, with each beetle host harbouring strongly differentiated assemblages of strains. Periodic out‐crossing generates admixture between genetically diverse lineages, creating a diverse array of allelic combinations likely to increase the evolutionary potential of the species and facilitate adaptation to local environments and beetle hosts.     

Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle

The deteriorating in vivo environment is thought to play a major role in reduced stem cell function with age. The capacity of stem cells to support tissue maintenance depends not only on their response to cues from the surrounding niche, but also on their abundance. Here, we investigate satellite cell (myogenic stem cell) pool size and its potential to participate in muscle maintenance through old age. The numbers and performance of mouse satellite cells have been analyzed using molecular markers that exclusively characterize quiescent satellite cells and their progeny as they transit through proliferation, differentiation and generation of reserve cells. The study establishes that abundance of resident satellite cells declines with age in myofibers from both fast- and slow-twitch muscles. Nevertheless, the inherent myogenic potential of satellite cells does not diminish with age. Furthermore, the aging satellite cell niche retains the capacity to support effective myogenesis upon enrichment of the mitogenic milieu with FGF. Altogether, satellite cell abundance, but not myogenic potential, deteriorates with age. This study suggests that the population of satellite cells that participate in myofiber maintenance during routine muscle utilization is not fully replenished throughout life.     

Antisense repression reveals a crucial role of the plastidic 2-oxoglutarate/malate translocator DiT1 at the interface between carbon and nitrogen metabolism

Ammonia assimilation by the plastidic glutamine synthetase/glutamate synthase system requires 2-oxoglutarate (2-OG) as a carbon precursor. Plastids depend on 2-OG import from the cytosol. A plastidic dicarboxylate translocator 1-[2-OG/malate translocator (DiT1)] has been identified and its substrate specificity and kinetic constants have been analyzed in vitro. However, the role of DiT1 in intact plants and its significance for ammonia assimilation remained uncertain. Here, to study the role of DiT1 in intact plants, its expression was antisense-repressed in transgenic tobacco plants. This resulted in a reduced transport capacity for 2-OG across the plastid envelope membrane. In consequence, allocation of carbon precursors to amino acid synthesis was impaired, organic acids accumulated and protein content, photosynthetic capacity and sugar pools in leaves were strongly decreased. The phenotype was consistent with a role of DIT1 in both, primary ammonia assimilation and the re-assimilation of ammonia resulting from the photorespiratory carbon cycle. Unexpectedly, the in situ rate of nitrate reduction was extremely low in alpha-DiT1 leaves, although nitrate reductase (NR) expression and activity remained high. We hypothesize that this discrepancy between extractable NR activity and in situ nitrate reduction is due to substrate limitation of NR. These findings and the severe phenotype of the antisense plants point to a crucial role of DiT1 at the interface between carbon and nitrogen metabolism.     

Gemcitabine functions epigenetically by inhibiting repair mediated DNA demethylation

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation.     

Accelerated biodegradation of cement by sulfur-oxidizing bacteria as a bioassay for evaluating immobilization of low-level radioactive waste

Disposal of low-level radioactive waste by immobilization in cement is being evaluated worldwide. The stability of cement in the environment may be impaired by sulfur-oxidizing bacteria that corrode the cement by producing sulfuric acid. Since this process is so slow that it is not possible to perform studies of the degradation kinetics and to test cement mixtures with increased durability, procedures that accelerate the biodegradation are required. Semicontinuous cultures of Halothiobacillus neapolitanus and Thiomonas intermedia containing thiosulfate as the sole energy source were employed to accelerate the biodegradation of cement samples. This resulted in a weight loss of up to 16% after 39 days, compared with a weight loss of 0.8% in noninoculated controls. Scanning electron microscopy of the degraded cement samples revealed deep cracks, which could be associated with the formation of low-density corrosion products in the interior of the cement. Accelerated biodegradation was also evident from the leaching rates of Ca(2+) and Si(2+), the major constituents of the cement matrix, and Ca exhibited the highest rate (up to 20 times greater than the control rate) due to the reaction between free lime and the biogenic sulfuric acid. Leaching of Sr(2+) and Cs(+), which were added to the cement to simulate immobilization of the corresponding radioisotopes, was also monitored. In contrast to the linear leaching kinetics of calcium, silicon, and strontium, the leaching pattern of cesium produced a saturation curve similar to the control curve. Presumably, the leaching of cesium is governed by the diffusion process, whereas the leaching kinetics of the other three ions seems to governed by dissolution of the cement.     

Head down tilt combined with breathing assistance by the "IRON LUNG." A new simulation model for cardiovascular deconditioning, skin, and kidney function in weightlessness?

In 1951 Gauer, Henry and Sieker proposed that "central hyper-volemia" might produce a diuresis that serves to reduce blood volume, a postulated negative-feedback system for the control of blood volume. Recent surprising results from human spaceflight indicate that although a central hyper-volemia takes place in weightlessness, an increase in salt and urine excretion cannot be observed. We hypothesised that on earth the mediastinum is shaped by gravity because of the gravity dependent hydrostatic gradient in the blood filled cavities and that its unloading by weightlessness reduces the wall stretch in the atria, thus resulting in reduced sodium and urine excretion. Therefore we have applied the principle of the "Iron Lung" (lung pressure manipulator LPM) in combination with a simulation method of weightlessness, head down tilt bed rest (HDT), to test this hypothesis. We found that similar to weightlessness, not only urinary excretion, but also evaporative water loss was reduced and that diastolic blood pressure increased.